Research ArticleVIRAL INFECTIONS

A durable protective immune response to wild-type measles virus infection of macaques is due to viral replication and spread in lymphoid tissues

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Science Translational Medicine  01 Apr 2020:
Vol. 12, Issue 537, eaax7799
DOI: 10.1126/scitranslmed.aax7799
  • Fig. 1 Lymphocyte counts and MeV N RNA in the blood and respiratory tract of infected macaques.

    MeV N–specific RT-PCR was used for detection and quantification of MeV N RNA after respiratory infection of macaques with either WT MeV or LAMV. (A) Number of macaques positive for MeV N RNA in nasal swab cell pellets from eight macaques 7 to 35 days after infection with WT MeV. (B) Quantification of MeV N RNA in bronchoalveolar lavage (BAL) fluids and lung tissues from 12 macaques 7 days after infection with LAMV. For quantification, MeV N RNA was normalized to the GAPDH control. Assays were performed in duplicate, and averaged results are expressed as (number of copies of MeV N RNA)/(number of copies of GAPDH RNA) × 5000. Numbers on the x axis identify individual macaques. (C) Comparison of the amounts of MeV N RNA in RNA extracted from 2 × 106 PBMCs from 12 LAMV-infected macaques or 8 WT MeV–infected macaques at 7, 14, 21, or 24 days after infection. P < 0.001. n.d., not detectable. (D) Quantification of MeV N RNA in the draining lymph nodes of 12 macaques 7 days after infection with LAMV. Numbers on the x axis identify individual macaques. (E) Numbers of lymphocytes × 1000/μl of peripheral blood after infection of individual macaques with WT MeV or LAMV. (F) The ratio of day 7 to day 0 lymphocyte counts for macaques infected with WT MeV (red circles) or LAMV (blue triangles). Significance was determined by Student’s t test, *P < 0.05.

  • Fig. 2 Macaque PBMC subsets infected by WT MeV during viremia.

    Flow cytometry was used to identify PBMCs expressing MeV N protein during viremia after intratracheal infection of macaques with 104 TCID50 of WT MeV. Representative flow cytometry images are shown for MeV N protein–expressing CD20+ B cells and CD3+ T cells from WT MeV–infected macaques 7 days after infection (A) and from uninfected control macaques (B). PE, phycoerythrin; APC, allophycocyanin. (C) Percentages of B cells (red circles) and T cells (blue squares) expressing MeV N protein 7 and 10 days after infection of macaques. Lines indicate the mean and SEM for each cell type and virus.

  • Fig. 3 Macaque T cell subsets infected by WT MeV during viremia.

    (A) Representative flow cytometry images show MeV N protein–positive CD4+ T cells (top) and CD8+ T cells (bottom) in the peripheral blood of macaques 7 days after infection with 104 TCID50 of the Bilthoven strain of WT MeV. Gating (red) is shown for naïve T cells, central memory (CM) T cells, and effector memory (EM) T cells (left), and MeV N protein–positive T cells in each group (right). (B) Quantification of the percentages of MeV N protein–positive CD4+ and CD8+ T cells that were central memory T cells, effector memory T cells, and naïve T cells for six individual macaques. Results from the same macaque are connected with lines. Significance was determined by ANOVA with Bonferroni post hoc tests. *P < 0.05, **P < 0.01.

  • Fig. 4 In vitro replication of WT MeV and LAMV in primary respiratory epithelial cells and PBMCs.

    Infectious MeV in apical supernatants and shedding of MeV-infected multinuclear giant cells were measured after apical or basal infection of primary differentiated macaque tracheal epithelial cells with LAMV or WT MeV (MOI = 4.5). (A) Apical cell culture supernatants (cultures from three separate macaques; two replicates in each experiment) were assayed for infectious virus by a TCID50 assay. Lines indicate the SEM. (B) Shed cells collected from macaque tracheal epithelial cell monolayers 144 hours after infection were stained with antibody to MeV N protein and DAPI nuclear stain after fixation and permeabilization. Merged images of phase-contrast, MeV N protein expression (green), and DAPI nuclear stain (blue) are shown. Scale bar represents 150 μm. (C to F) Production of infectious virus 24 hours after infection of human PBMCs with WT MeV (red circles) and LAMV (blue triangles) at high (2 to 5) MOI (C) and low (0.5) MOI (D). Production of infectious virus 24 hours after infection of human CD4+ T cells (E) or CD14+ myeloid cells (F) with WT MeV (red circles) or LAMV (blue triangles) (MOI = 5). Horizontal line indicates the mean. Significance was determined by Mann-Whitney U test. *P < 0.05 and **P < 0.01. ns, not significant.

  • Fig. 5 In vitro replication of WT MeV and LAMV in human primary CD4+ T cells.

    Shown is flow cytometry analysis of MeV N protein expression after in vitro infection of human PBMCs with WT MeV or LAMV (vaccine strain of MeV). (A) Flow cytometry histogram shows amounts of MeV N protein expressed by human CD4+ T cells 20 hours after infection with WT MeV or LAMV at an MOI of 0.5 or 5.0. (B and C) Flow cytometry plots of CD4+ T cell expression of MeV N protein 20 hours after infection with LAMV (B) or WT MeV (C) strains (MOI = 5.0). SSC, side-scattered light. (D and E) Each panel shows results of three separate flow cytometry experiments that assessed the percentage of human CD4+ T cells expressing MeV N protein (D) and the amount of MeV N protein measured by immunofluorescence (E) 48 hours after infection of human PBMCs with WT MeV or LAMV (MOI = 5). Lines connect data from the same experiment.

  • Fig. 6 Plasma cytokine and chemokine concentrations after WT MeV or LAMV infection of macaques.

    Plasma concentrations of IL-1β, IFN-γ, IL-12, CCL2, CCL11, CCL22, CXCL9, CXCL11, MIF, and βFGF were measured using a Luminex 28-plex monkey cytokine/chemokine assay panel. Plasma samples were run in duplicate for five animals infected with WT MeV (blue) or with LAMV (orange). Data are plotted as means ± SEM. Significance of differences between groups was determined by two-way ANOVA using data from days 0, 7, 14, and 24 or 28 after infection. For analysis of IFN-γ, the two highest outliers (both in the WT MeV–infected group) were removed.

  • Fig. 7 MeV-specific antibody responses after WT MeV or LAMV infection of macaques.

    Plasma concentrations of (A) MeV-specific IgG, (B) MeV-specific IgA, and (C) H-specific IgG were measured by enzyme immunoassay (EIA) using plates coated with a lysate from MeV-infected Vero cells or MeV H–expressing L cells. Titers of antibodies were determined on the basis of the curve of serially diluted plasma and assay-specific background values. (D) Reciprocal titers of neutralizing antibody in macaque plasma samples were determined by a plaque reduction neutralization test (PRNT) using the Chicago strain of MeV and Vero cells. Results were calculated from assay triplicates. (E) Avidity of MeV-specific IgG was determined in triplicate by an EIA-based assay and expressed as avidity index (AI) for six individual macaques at 14 weeks after infection with WT MeV or LAMV. (F) MeV-specific IgG-secreting cells in the bone marrow were assessed by ELISpot assay to identify the numbers of cells secreting antibody to MeV. Shown are representative wells loaded with 5 × 105 bone marrow cells from macaques approximately one year after infection with WT MeV (35 and 28 spots) or LAMV (2 and 3 spots). Spots represent MeV-specific IgG-secreting long-lived plasma cells (LLPCs). (G) Number of MeV-, H-, and N-specific IgG-secreting long-lived plasma cells in macaque bone marrow assessed by ELISpot assay about 1 year after infection. Averaged number of spots from eight assay replicates in individual animals was plotted. *P < 0.05, **P < 0.01, and ***P < 0.001. Data in (A) and (B) were compared using two-way ANOVA with Bonferroni post hoc tests; data in (C), (D), (E), and (G) were compared using Student’s t test.

  • Fig. 8 Cellular immune responses after WT MeV and LAMV infection of macaques.

    Numbers of IFN-γ– and IL-4–producing T cells in infected macaques were assessed by ELISpot assay. Fresh macaque PBMCs (5 × 105) were added to multiscreen plates coated with anti-human IFN-γ or IL-4 antibody in the presence of pooled H or N peptides or medium. Data are reported as spot-forming cells (SFCs) after subtracting medium alone wells from antigen-stimulated wells. (A) Numbers of H- and N-specific IFN-γ–producing cells 14 and 70 days postinfection (dpi). (B) Numbers of N-specific IL-4–producing cells 14 days after infection. Horizontal line indicates the mean. (C) Changes in numbers of N-specific IFN-γ–producing cells in the circulation of infected macaques over the course of MeV infection. Reactivation of MeV-specific T cells in the circulation was noted around 60 days after infection for animals infected with WT MeV (red circles) or LAMV (blue triangles).

Supplementary Materials

  • This PDF file includes:

    • Fig. S1. Amino acid sequence alignment for the EZ vaccine strain (LAMV) and Bilthoven strain of WT MeV (ViPR; www.viprbrc.org/).
    • Table S1. Individual-level data for Fig. 6.
    • Table S2. Individual-level data for Fig. 7A.
    • Table S3. Individual-level data for Fig. 7C.

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